Despite the implementation of enhanced recovery protocols for GI surgeries, there is still significant postoperative ileus (POI) and postoperative gastrointestinal dysfunction (POGD) associated with prolonged hospitalizations, increased morbidity and health care costs into the billions. Novel therapies in the pipeline are limited. A better understanding of the pathogenic mechanism(s) of POI is required to develop better therapeutic strategies. Emerging evidence indicates that enteric glia play an important role in health and disease of the gastrointestinal tract. Endothelin (ET-1) signaling'` in `reactive enteric glia' in the gut is a novel target of investigation for us in the pathogenesis of POI and POGD (Gulbrensan & Christofi, Gastro, 2018). Our recent work, strong pilot, feasibility and preliminary data (Figs 1-16), abstracts and publications support the novel hypothesis that glial ET-1 / ETBR signaling in enteric glia disrupts motility and it contributes to the pathogenesis of POI and POGD in the context of intestinal inflammation. Pathogenic mechanism(s) involving endothelins include glial hypersensitivity to ET-1/ETBR signaling, induction of a reactive glial phenotype, enteric gliosis, enteric neuropathy, and alterations in neural-motor pathways. Our study will also investigate the therapeutic potential of targeting ETBR in POI and POGD. We will test our general hypothesis in 3 specific aims in mice and humans: Aim 1 will focus on the Mechanism of ET-1/ETBR inhibition of motility. A multi-tier approach is used to study ETBR on motility in mouse and human, including muscle contraction, peristaltic waves, pellet propulsion, ascending and descending reflexes and GI transit. ETBR signaling in glia, neurons and motility is studied in cell-specific Ca2+ reporter mice for glia, neurons, excitatory and inhibitory neuron pathways, transgenic models of glial conditional ETBR ablation or ET-1 up regulation, and ETBR-EGFP reporter mice. Translatability of findings from mice to humans is tested on Ca2+waves in human EGC networks, `first in human' patch clamp recordings in networks of myenteric ganglia', and ETBR studies of ascending and descending reflexes. Aim 2 will focus on the pathogenic mechanism of ETBR signaling in enteric glia in POI ? Pathogenic mechanisms include hypersensitivity in ET-1/ETBR signaling, induction of a reactive glial phenotype analyzed by RNAseq in RiboTag mice, and mice generated by crossing RiboTag with glial-specific ETBR knockouts. Translatability of findings to humans is tested in hEGCs or networks of ganglia with Ca2+or patch clamp recordings. Impact on enteric neuropathy is tested in mice with glial ablation of ETBR or ET-1 up regulation in Ca2+reporter mice for neurons. Aim 3 will focus on glial ET-1 and ETBR as a therapeutic target in POI using a gut surgical manipulation model of POI and colectomy patients with POI, and pharmacological antagonists in mice, or testing in mice with glial ablation of ETBR or up regulation of ET-1. Overall, a novel glial ET-1 / ETBR pathogenic mechanism of postoperative ileus may be identified as a relevant therapeutic target for prophylactic treatment.